The T-cell receptor is essential for major functions of the adaptive immune system and is one of the most complex cell surface receptors. It is composed of eight polypeptide chains that must be assembled in the ER in the proper stoichiometry for it to perform its vital functions. As such, its assembly poses a formidable task for the ER quality control machinery. Although a large body of literature exists on the assembly and quality control of the TCR, these efforts have focused primarily on the unusual transmembrane domains of the individual chains. These possess charged residues that, when unpaired, accelerate degradation of the TCR chains and are believed to drive assembly. However, the basis of this presumed integral membrane quality control step has not been elucidated. Furthermore, very little attention has been directed to possible roles for the lumenal portions of this receptor, which are likely to be the regions scrutinized by the known quality control machinery of the ER. To remedy this deficiency, we propose to combine biophysical and cell based studies to obtain high resolution structural and kinetic data on the folding and assembly of the TCR that can be correlated with checkpoints in the cell. Our preliminary data obtained from these approaches have already revealed two unanticipated features of the TCR -chain. We find that its constant domain is unstructured in the absence of association with the - chain and that its transmembrane region is not integrated into the ER membrane when expressed alone. These two features are very likely to provide checkpoints in the quality control of receptor assembly. These preliminary insights will be expanded in order to obtain an integrated view of the ER mechanisms that aid and monitor TCR biosynthesis allowing only properly assembled receptors to be expressed on the cell surface. PUBLIC HEALTH RELEVANCE: Although there has been much progress in identifying components of the ER quality control machinery, the underlying mechanisms for executing them remain poorly understood. This is particularly true for transmembrane proteins that control important functions in multicellular organisms and are often pharmacological targets. We propose studies to decipher novel aspects in the assembly and quality control of the essential T cell antigen receptor (TCR), one of the most complex cell surface receptors known. We chose the TCR because strict quality control checkpoints are known to scrutinize the assembly of this eight-chain receptor, yet the underlying molecular mechanisms are not well understood. The role of its lumenal domains in quality control has not been adequately investigated, and a putative transmembrane quality control step has remained vague. Both layers of quality control will be addressed within our proposed project. The mechanistic insights gained from our proposed combination of in vitro and in vivo techniques have the potential to contribute to the development of more direct methods for assessing protein folding in the cell, which is currently limited to indirect methods like disulfide bond formation, interactions with molecular chaperones, and further transport along the secretory pathway versus degradation. In addition to increasing our general understanding of ER quality control, our study will provide novel insights into the biosynthesis of TCR that will likely apply to other immunoreceptors and might provide novel ways to manipulate them therapeutically.